Competition between Der1 and ERAD-M substrates controls Hrd1 complex function.

Endoplasmic reticulum-associated degradation (ERAD) is a quality control process which removes misfolded proteins from the ER. The central component of the most conserved ERAD system is an integral membrane ubiquitin ligase called Hrd1. The Hrd1 ligase functions within a complex to mediate the recognition and ubiquitination of both soluble, lumenal substrates and integral membrane substrates, all of which are ultimately targeted for degradation by the cytosolic proteasome. Here, we used deep mutational scanning to identify Hrd1 residues exclusively involved in the degradation of integral membrane substrates. We report single residue Hrd1 variants that are broadly deficient in the degradation of all integral membrane substrates tested. Using in vivo assays to characterize Hrd1 variant deficiency, we explain how integral membrane substrates compete with other complex components to control Hrd1 function. This work reveals competition for the retrotranslocon cavity between both lumenal and membrane substrate degradation paths and highlights Hrd1 complex assembly as the primary determinant for tuning ERAD function.